An outwardly rectifying anionic background current in atrial myocytes from the human heart.

This report describes a hitherto unreported anionic background current from human atrial cardiomyocytes. Under whole-cell patch-clamp with anion-selective conditions, an outwardly rectifying anion current (I(ANION)) was observed, which was larger with iodide than nitrate, and with nitrate than chloride as charge carrier. In contrast with a previously identified background anionic current from small mammal cardiomyocytes, I(ANION) was not augmented by the pyrethroid tefluthrin (10 microM); neither was it inhibited by hyperosmolar external solution nor by DIDS (200 microM); thus I(ANION) was not due to basal activity of volume-sensitive anion channels. I(ANION) was partially inhibited by the Cl(-) channel blockers NPPB (50 microM) and Gly H-101 (30 microM). Incorporation of I(ANION) into a human atrial action potential (AP) simulation led to depression of the AP plateau, accompanied by alterations to plateau inward calcium current, and to AP shortening at 50% but not 90% of complete repolarization, demonstrating that I(ANION) can influence the human atrial AP profile.

Endothelial cells freshly isolated from small pulmonary arteries of the rat possess multiple distinct k+ current profiles.

This study demonstrates for the first time that endothelial cells freshly isolated from small pulmonary arteries of the rat, based on their electrophysiological profile, possess two distinct populations of cells. Immunohistochemical staining revealed the presence of both anti-Kv1.5 and anti-Kir2.1 immunoreactivity in the endothelium of small pulmonary arteries. Patch-clamp studies demonstrated that 90% of cells studied exhibited an electrophysiological profile that was characterized by a delayed rectifier K+ conductance. However, the remaining 10% of cells studied showed the complete absence of a delayed rectifier K+ current and were characterized by an inward rectifier K+ conductance. Together these results indicate that endothelial cells isolated from rat small pulmonary arteries possess a heterogeneous population of cells that may be distinguished by their markedly different electrophysiological profiles. These different populations of cells may differ in their control of the resting membrane potential of endothelial cells, and thereby altering Ca2+ homeostasis and release of vasoactive compounds. These findings may therefore have important implications for understanding the regulation of pulmonary vascular tone.

Actions of pyrethroid insecticides on sodium currents, action potentials, and contractile rhythm in isolated mammalian ventricular myocytes and perfused hearts.

Pyrethroid insecticides are known to modify neuronal sodium channels, inducing persistent, steady-state sodium current at depolarized membrane potentials. Cardiac myocytes are also rich in sodium channels but comparatively little is known about the effect of pyrethroids on the heart, or on the cardiac sodium channel isoform. In the present study therefore, we determined the actions of type I and type II pyrethroids against rat and guinea pig ventricular myocytes under current and voltage clamp, and on isolated perfused rat hearts. In myocytes, tefluthrin (type I) and fenpropathrin and alpha-cypermethrin (type II) prolonged action potentials and evoked afterdepolarizations. The time course of sodium current (I(Na)) was also prolonged by these compounds. Pyrethroids delayed I(Na) inactivation, when measured under selective conditions as current sensitive to 30 microM tetrodotoxin, by increasing the proportion of slowly inactivating current at the expense of fast inactivating current. Further experiments, focusing on fenpropathrin, revealed that its effects on I(Na) inactivation time course were dose-dependent, and the Na(+) "window-current" was increased in its presence. In unstimulated, isolated hearts perfused with the same pyrethroids, the variability in contraction amplitude increased due to variations in the intervals between heartbeats. These potentially arrhythmogenic changes are consistent with the effects observed at the cellular level. The type I pyrethroid tetramethrin had little effect in any of the preparations. These findings suggest that some pyrethroids possess considerable mammalian cardiac arrhythmogenic potential, the manifestation of which in vivo may depend on the route of exposure.

Open day review: bridging the gap between academia and industry.

In conclusion, the meeting was viewed positively by academics, industrialists and administrators. The scientists generated considerable interest with some positive follow-up. Above all, it was clear that Bristol University is working to support the bridge that already spans the gap between academic and industrial pharmacology.

Kv channel subunit expression in rat pulmonary arteries.

Hypoxic pulmonary vasoconstriction (HPV) is a mechanism whereby capillary perfusion is modulated to match alveolar ventilation by diverting blood flow away from poorly ventilated regions of the lung. K+ channels, sensitive to changes in oxygen tension, are thought to play a pivotal role in initiating contraction of pulmonary arterial smooth muscle cells. However, the specific channel subtypes involved have not yet been identified. Using RT-PCR, we have investigated the expression of delayed rectifying (Kv) channel mRNA in rat main and small pulmonary arteries and, for comparison, the systemic mesenteric artery. We have identified and fully sequenced a rat Kv9.2 cDNA and also demonstrated the presence of Kv1.7 and Kv4.1. The presence and relative distribution of Kv1.2, Kv1.5, Kv2.1, and Kv9 mRNA is consistent with the proposed contribution of these subunits to oxygen sensing by K channels, previously described in pulmonary arteries. Our data addresses the controversy relating to the likely distribution of Kv channels involved in oxygen sensing without necessarily implying that such subunits are directly responsible for this process. The differential expression of other subunits, particularly Kv4, indicates that these too may have a role in HPV, revealing the need for further biophysical evaluation of these channel subtypes.

Electrophysiological effects of endothelin-1 and their relationship to contraction in rat renal arterial smooth muscle.

The electophysiological effects of endothelin-1 (ET-1) and their relationship to contraction remain unclear in the renal circulation. Using endotheliumdenuded arteries from the main branch of the renal artery proximal to the kidney of the rat, we have examined its effects on tension and conducted parallel patch-clamp measurements using freshly isolated smooth muscle cells from this tissue. Pharmacological experiments revealed that ET-1 produced constriction of renal arteries dependent on the influx of extracellular Ca(2+), mediated solely through ET(A) receptor stimulation. Current-clamp experiments revealed that renal arterial myocytes had a resting membrane potential of approximately 32 mV, with the majority of cells exhibiting spontaneous transient hyperpolarizations (STHPs). Application of ET-1 produced depolarization and in those cells exhibiting STHPs, either caused their inhibition or made them occur regularly. Under voltage-clamp conditions cells were observed to exhibit spontaneous transient outward currents (STOCs) inhibited by iberiotoxin. Application of voltage-ramps revealed an outward current activated at approximately -30 mV, sensitive to both 4-AP and TEA. Taken together these results suggest that renal arterial myocytes possess both delayed rectifying K(+) (K(V)) and Ca(2+)-activated K(+) (BK(Ca)) channels. Under voltage-clamp, ET-1 attenuated the outward current and reduced the magnitude and incidence of STOCs: effects mediated solely as a consequence of ET(A) receptor stimulation. Thus, in conclusion, activation of ET(A) receptors by ET-1 causes inhibition of K(V) and BK(Ca) channel activity, which could promote and/or maintain membrane depolarization. This effect is likely to favour L-type Ca(2+) channel activity providing an influx pathway for extracellular Ca(2+) essential for contraction.

A novel anionic conductance affects action potential duration in isolated rat ventricular myocytes.

Effects of extracellular anions were studied in electrophysiological experiments on freshly isolated rat ventricular myocytes. Under current-clamp, action potential duration (APD) was prolonged by reducing the extracellular Cl(-) concentration and shortened by replacement of extracellular Cl(-) with I(-). Under voltage-clamp, membrane potential steps or ramps evoked an anionic background current (I(AB)) carried by either Cl(-), Br(-), I(-) or NO(3)(-). Activation of I(AB) was Ca(2+)- and cyclic AMP-independent, and was unaffected by cell shrinkage. I(AB) was insensitive to stilbene and fenamate anion transport blockers at concentrations that inhibit Ca(2+)-, cyclic AMP- and swelling-activated Cl(-) currents in ventricular cells of other mammals. These results suggest that I(AB) may be carried by a novel class of Cl(-) channel. Correlation of anion substitution experiments on membrane current and action potentials revealed that I(AB) could play a major role in controlling rat ventricular APD. These findings have important implications for those studying cardiac Cl(-) channels as potential targets for novel antiarrythmic agents.

Signature currents: a patch-clamp method for determining the selectivity of ion-channel blockers in isolated cardiac myocytes.

BACKGROUND: We describe a simple method using membrane potential ramps for rapidly determining the ion-channel selectivity of drugs that affect action-potential duration in isolated cardiac myocytes. The method allows the simultaneous assay of compounds on a number of ionic currents in a single cardiac cell. METHODS: Trains of membrane potential ramps were applied from -90 to +70 mV at 0.33 Hz to obtain a consistent "signature current," in which the major individual currents involved in the cardiac action potential could be easily identified. Confirmatory experiments were performed using known inhibitors of these currents. RESULTS: The identities of the currents in the signature were established by varying the concentrations of extracellular cations and by adding known ion channel blockers to superfusion solutions. Inhibition of each current had a characteristic and reproducible effect on the overall signature current. CONCLUSIONS: The consistent current signature in the presence and absence of blockers suggests that this method could be used for tertiary electrophysiological evaluation of compounds, eg, in a drug discovery program focusing on antiarrhythmic agents. The ability to assay for secondary effects of novel compounds against multiple currents in the target cell type is convenient and avoids the artefacts associated with using artificial expression systems.

Endothelial cells freshly isolated from resistance-sized pulmonary arteries possess a unique K(+) current profile.

We have, for the first time, developed a reliable method for freshly isolating viable endothelial cells from resistance-sized rat pulmonary arteries. The endothelial origin of these cells was confirmed using indirect immunofluorescence, utilizing fluorescently labeled low-density lipoprotein. Biophysical and pharmacological patch-clamp experiments conducted under quasiphysiological cationic gradients revealed that these cells had a mean resting membrane potential of approximately -38 mV and displayed a delayed-rectifying K(+) current. Immunohistochemical staining of rat lung cross-sections revealed an abundance of K(V)1.5 channel protein in pulmonary endothelium. This is the first report of a delayed-rectifying K(+) current in endothelial cells of resistance-sized pulmonary arteries. Since changes in membrane potential associated with K(+) channel activity affect release of vasoactive substances from endothelial cells, this finding has important implications for understanding the mechanisms underlying control of pulmonary vascular tone.

Relaxation of endothelin-1-induced pulmonary arterial constriction by niflumic acid and NPPB: mechanism(s) independent of chloride channel block.

We investigated the effects of the Cl- channel blockers niflumic acid, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and 4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) on endothelin-1 (ET-1)-induced constriction of rat small pulmonary arteries (diameter 100-400 microm) in vitro, following endothelium removal. ET-1 (30 nM) induced a sustained constriction of rat pulmonary arteries in physiological salt solution. Arteries preconstricted with ET-1 were relaxed by niflumic acid (IC50: 35.8 microM) and NPPB (IC50: 21.1 microM) in a reversible and concentration-dependent manner. However, at concentrations known to block Ca++-activated Cl- channels, DIDS (

Functional evidence for a novel suramin-insensitive pyrimidine receptor in rat small pulmonary arteries.

Uridine nucleotides are known to cause constriction of pulmonary arterial smooth muscle. However, the P2 receptor subtypes underlying the contractile effects of these nucleotides in the pulmonary circulation have not been determined. We have used myography and the patch-clamp recording technique to compare the effects of UTP and UDP in isolated small pulmonary arteries (diameter 100 to 400 microm) and their constituent smooth muscle cells. In endothelium-denuded arteries, both UTP and UDP (0.01 to 3 mmol/L) induced concentration-dependent increases in tension that were independent of P2X receptor stimulation. The UDP-mediated increase in tension was significantly less sensitive to the nonselective P2 receptor blocker suramin than the UTP-mediated increase in tension. In single isolated arterial myocytes, voltage-clamped at -50 mV (close to the resting membrane potential of these cells), application of both UTP and UDP evoked periodic oscillations of inward current primarily because of a Ca2+-activated Cl- current (ICl,Ca). Oscillations of ICl,Ca evoked by UTP were reversibly inhibited by suramin, although those evoked by UDP were insensitive to the antagonist. In addition to confirming the presence of classical P2Y2 receptors, these results also provide functional evidence for the existence of a novel UDP receptor in pulmonary arterial myocytes, which may contribute to pyrimidine-evoked vasoconstriction. This notion is supported by molecular evidence that demonstrates the presence of P2Y6 receptor transcripts in rat pulmonary arterial smooth muscle.

Differential effects of hypoxia on the intracellular Ca2+ concentration of myocytes isolated from different regions of the rat pulmonary arterial tree.

Hypoxic pulmonary vasoconstriction (HPV) may be mediated, in part, by an oxygen-sensing mechanism intrinsic to pulmonary arterial smooth muscle. It has been proposed that hypoxia inhibits a K+ conductance, which promotes membrane depolarization, subsequent activation of L-type Ca2+ channels and ultimately constriction. We have monitored hypoxia-induced changes in the intracellular Ca2+ concentration ([Ca2+]i) of single myocytes isolated from the rat pulmonary arterial tree using microspectrofluorimetry and ratiometric measurement of indo-1 fluorescence. The basal level of [Ca2+]i was in the range 35-80 nM and cells were quiescent at rest exhibiting no spontaneous oscillations in [Ca2+]i. When the extracellular K+ concentration ([K+]o) was raised to 20 mM, the [Ca2+]i increased from approximately 60 to approximately 100 nM. This increase was abolished by nifedipine, demonstrating the presence, and need for activation, of functional voltage-gated L-type Ca2+ channels. Hypoxia (PO2 < or = 30 mmHg; throughout) had little effect on the resting [Ca2+]i in myocytes isolated from either the main intrapulmonary artery, or its primary, secondary or tertiary branches. However, upon raising the [Ca2+]i by increasing [K+]o to 20 mM, hypoxia was found to lower [Ca2+]i from approximately 110 to approximately 70 nM, in cells isolated from the main conduit and primary branches of the intrapulmonary artery. In marked contrast, when [Ca2+]i was raised, by increasing [K+]o to 20 mM, in myocytes isolated from secondary and tertiary branches of the intrapulmonary artery, hypoxia induced a further reversible increase in the [Ca2+]i from approximately 160 to approximately 240 nM. Neither hypoxia alone nor in combination with 20 mM K(+)o induced any increase in the [Ca2+]i in the presence of nifedipine. We conclude that hypoxia may modulate [Ca2+]i in rat pulmonary artery myocytes only following its elevation by a depolarizing stimulus.

Endothelin-1, delayed rectifier K channels, and pulmonary arterial smooth muscle.

Sarafotoxin S6c [STXS6c; a selective endothelin-B (ETB) receptor agonist] causes constriction of isolated pulmonary arteries. In perforated-patch experiments on pulmonary arterial myocytes, ET-1 and STXS6c induced a gradual inhibition of the delayed rectifier K current (IKV), the profile of which resembled that carried by Kv1.5. Reverse-transcriptase polymerase chain reaction (RT-PCR) experiments revealed mRNA encoding this channel, and immunolocalization experiments demonstrated expression of the channel protein in pulmonary arterial smooth muscle. It is tempting to speculate that ETB receptor coupling to Kv1.5 may be implicated in contraction after stimulation of these receptors.

Differential electrophysiological actions of endothelin-1 on Cl- and K+ currents in myocytes isolated from aorta, basilar and pulmonary artery.

The electrophysiological effects of endothelin (ET)-1 were compared in myocytes isolated from rat small pulmonary artery, basilar artery and aorta. ET-1 evoked depolarization in all three smooth muscle cell types. Depolarizing oscillations in membrane current also were observed in pulmonary and aortic myocytes. In voltage-clamp experiments ET-1 induced a gradual inhibition of the Ca(++)-independent outward current (IK) in pulmonary and aortic myocytes, whereas in basilar myocytes ET-1 inhibited the Ca(++)-activated K+ current (IK(Ca)). ET-1 also evoked a transient enhancement of IK(Ca) and oscillations in inward current in aortic and pulmonary myocytes. The inward currents were inhibited by caffeine, which suggests Ca(++)-dependent activation. Ion-exchange experiments indicated that in pulmonary myocytes oscillatory currents were caused solely by the movement of Cl-, whereas in aortic myocytes they were the consequence of both Ca(++)-activated Cl-(ICl(Ca)) and non-selective cation currents (INS). No inward current was evoked in basilar myocytes in response to ET-1 or photorelease of Ca++, which suggests that these cells do not possess ICl(Ca). Experiments with ET receptor ligands indicated that in basilar myocytes ETA receptor stimulation is responsible for IK(Ca) inhibition, whereas in aortic and pulmonary myocytes ETB and ETA receptor stimulation mediates inhibition of IK and activation of ICl(Ca), INS and IK(Ca), respectively. In the future, it may be possible to exploit these differential effects of ET-1 pharmacologically to assist development of tissue-specific modulators for the treatment of vascular disease.

Relationship between membrane potential, delayed rectifier K+ currents and hypoxia in rat pulmonary arterial myocytes.

Pulmonary arteries constrict in response to hypoxia, a process thought to involve oxygen sensing by K+ channels. We therefore investigated the effects of hypoxia on voltage-activated K+ currents in myocytes isolated from rat small pulmonary arteries using the patch-clamp recording technique. Experiments with iberiotoxin and intracellularly applied Ca2+ chelating agents revealed that hypoxia (PO2, 20-30 mmHg; throughout) inhibited the Ca(2+)-insensitive component of the delayed voltage-activated outward K+ current. Hypoxia did not affect the membrane potential of these cells until they were depolarized by extracellular application of 20 mM K+, current injection or endothelin-1. Hypoxia caused little depolarization in the presence of prostaglandin F2 alpha, an agonist which was ineffective at inducing depolarization. These results suggest that an initial 'priming' depolarization may confer a sensitivity to hypoxia by activating delayed rectifier (Kv) channels. Once active, these channels can then be closed by hypoxia, leading to further depolarization. It is unlikely, therefore, that Kv channels are involved in controlling the resting membrane potential of these cells.

Electrophysiological consequences of purinergic receptor stimulation in isolated rat pulmonary arterial myocytes.

Neither the electrophysiological effects of purinergic receptor stimulation nor the role of ATP in regulating the tone of pulmonary arterial smooth muscle has been determined. Therefore, we investigated the effects of purine nucleotides on acutely dissociated smooth muscle cells from rat small pulmonary arteries using the patch-clamp recording technique. Extracellular application of ATP activated a fast transient inward current (which decayed in the continued presence of the nucleotide) and produced sustained periodic oscillations of predominantly inward current. Pharmacological and anion substitution experiments revealed that the transient inward current was carried by the movement of cations. In contrast, the periodic oscillations of current were due primarily to a Ca(2+)-activated Cl- current (ICl,Ca) dependent on the release of Ca2+ from intracellular stores. Experiments using ATP analogues revealed the following order of potency for activation of the fast transient inward current: 2-methylthio ATP (2-meSATP) > ATP > alpha,beta-methylene ATP (alpha,beta-meATP) > > ADP > UTP = adenosine. Cross desensitization was seen between applications of ATP, alpha,beta-meATP, and 2-meSATP, suggesting that these agonists act via a common site. The order of potency for activation of ICl,Ca was UTP = ATP > > ADP > or = 2-meSATP > alpha,beta-meATP = adenosine. Both the fast transient inward current and ICl,Ca evoked by ATP and its analogues were abolished by the nonselective P2 purinoceptor antagonist suramin. These results show the existence of P2x and P2U purinoceptor subtypes in pulmonary arterial smooth muscle cells. Stimulation of these receptors results in activation of a fast transient inward cation current and ICl,Ca, respectively. It is likely that ATP acts via these receptor subtypes to regulate pulmonary arterial tone under physiological or pathological conditions.

Endothelin receptor coupling to potassium and chloride channels in isolated rat pulmonary arterial myocytes.

Endothelin (endothelin)-1 may in important role in the control of pulmonary arterial tone, part of its action being due to a putative ability to regulate membrane ion channel activity. Consequently, we have examined its effects on membrane currents in pulmonary arterial myocytes by using the patchclamp recording technique. Endothelin-1 (0.1-50 nM) activated an oscillatory inward current and caused a brief enhancement, followed by inhibition of the outward K+ current. Pharmacological and anion substitution experiments revealed that the oscillations of inward current were due to ET(A) receptor stimulation and subsequent activation of Ca(+2)-activated Cl- channels (EC50 > or = 16 nM). Enhancement of K+ current was inhibited by FR139317 (an ET(A) receptor antagonist) and by buffering intracellular Ca+2, which suggests that ET(A) receptor stimulation also activates Ca(+2)-activated K+ channels. Inhibition of K+ current (IC50 approximately 0.5 nM) by endothelin-1 was not dependent on phosphorylation or intracellular Ca+2 and was unaffected by FR139317 but was mimicked by ET(B) receptor agonists. It appears, therefore, that ET(A) and ET(B) receptors in pulmonary arterial myocytes are differentially coupled to Cl- and K+ channels. Modulation of these channels by endothelin-1 may prove to play an important role in regulating pulmonary arterial smooth muscle tone under both physiological and pathological conditions.

Contractile agonists preferentially activate CL- over K+ currents in arterial myocytes.

Simultaneous patch-clamp and Ca2+ fluorescence measurements have revealed depolarising oscillations in the membrane potential of arterial (pulmonary) myocytes in response to adenosine 5'-triphosphate (ATP) and endothelin-1 (ET-1). These oscillations (i) are due to the preferential activation of Ca(2+)-activated Cl-, over K+ currents, (ii) occur through a mechanism involving Ca2+ release from intracellular Ca2+ stores and (iii) are likely to promote constriction. These results provide a novel perspective into the relative contribution and importance of Ca2+ activated Cl- and K+ channels in controlling membrane potential of arterial smooth muscle in response to contractile agonists.

ATP increases Ca(2+)-activated K+ channel activity in isolated rat arterial smooth muscle cells.

Large conductance Ca(2+)-activated K+ (Kca) channels are known to be activated by phosphorylation through cAMP- and cGMP-dependent kinase activation. In pulmonary arterial smooth muscle KCa channels are directly activated by ATP (but not by non-hydrolysable analogues) independently of the presence of cyclic nucleotides or the catalytic subunits of protein kinases. This study was designed to determine whether direct activation of KCa channels by ATP is apparent in other types of arterial smooth muscle. KCa channels of similar conductance to those of rat pulmonary artery (approximately 250 pS) were found in membrane patches excised from isolated smooth muscle cells from rat aorta, mesenteric and basilar arteries. In myocytes isolated from each of these arteries, intracellular application of ATP (in the absence of exogenous cyclic nucleotides or catalytic subunits) reversibly increased the open state probability of KCa channels: a response markedly reduced by a specific inhibitor of protein kinase A. Nucleotide sequence analysis of KCa channels revealed no homology with the majority of protein kinases. It is concluded that phosphorylation of KCa channels through the activity of a membrane tethered kinase related to protein kinase A (but lacking its regulatory subunits) may play an important role in controlling K+ flux in a range of arterial smooth muscle cell types.

Ca(2+)-activated Cl- currents in pulmonary arterial myocytes.

Currents from smooth muscle cells isolated from the pulmonary arterial tree of the rat were recorded under voltage clamp using the whole cell configuration of the patch-clamp technique. Rapid increases in cytosolic free calcium evoked by flash photolysis of Nitr-5 activated a current that, following ion substitution and pharmacological experiments, proved to be carried by Cl-. This current [ICl(Ca)] was evoked independently of photolytic by-products and, although smaller, was still activated in the absence of pipette ATP. Experiments revealed that ICl(Ca) was evoked in 80% in the cells isolated from the main pulmonary artery but only in 43% of the cells isolated from small vessels (200-400 microns ID). Application of caffeine also resulted in activation of ICl(ca), although the response current magnitude was larger in the main pulmonary artery. Photolysis of Nitr-5 still activated ICl(ca) in the presence of caffeine, suggesting that Ca2-release is not a prerequisite for activation of ICl(ca). These results represents in the first electrophysiological recordings of Cl- currents from small pulmonary arterial vessels and indicate that their Ca2+ regulation and/or distribution may be different throughout the pulmonary circulation.